At the present time, the only available “assays” based on cognitive or “cellular function” are living creatures. The use of C. elegans and Drosophila is predominant in these assays. In vitro cell cultures of embryonic rat and mouse tissue, especially through “knockout” technology, have also been used to study cellular organization and communication. However, in studies to date, single neurons have not been electrically integrated with modern electronics except in expensive patch-clamp methodology, which can be tedious, can result in disorganized cultures and are incapable of high throughput analysis. Imaging capabilities have been introduced using voltage-sensitive dyes. Such techniques are limited in their use, however, and dyes are generally toxic to neurons, as already mentioned above.
Therefore, it is clear that there is a lack of in vitro assays for studying neurotoxicity, for example, which are based on a cell's function. There are also very few methods of measuring toxicity other than morphological analysis. There is also a problem performing chronic electrophysiological monitoring of cells by standard electrophysiology. What is more the field of genomics lacks a high throughput assay to analyze the large number of sequences and genes being uncovered by the human genome project. Thousands upon thousands of new compounds are also being generated by recently utilized combinatorial chemical synthesis methods.
The use of biological cells and their underlying cellular functions as model systems for information processing is being investigated for a number of practical applications. Algorithms that are based on olfactory processing (Ambrose-Ingerson, J., Granger, R., and Lynch, G. (1990). Science 247: 1344-1348.; Granger, R., Ambrose-Ingerson, J., Anton, P. S., Whitson, J., and Lynch, G. (1991). An Introduction to Neural and Electronic Networks, eds. Zornetzer, S. F., Davis, J. L., and Lau, C. (Academic Press, Inc., San Diego), pp. 25-42.), computing using DNA in a test tube (Adleman, L. (1994). Molecular Computation of Solutions to Combinatorial Problems. Science Vol. 266.), or possibly manipulation of DNA in bacteria or other cells are just some examples. Recent articles have focused on bioinformation and the creation of biological pathways or genetic circuits using silicon-based models (Palsson, 1997). Experiments on tissue slice preparations, in cultured neuronal networks (Gross, G. W., Rhoades, B. K., Azzazy, H. M. E., & Wu, M. C. (1995). The use of neuronal networks on multielectrode arrays as biosensors. Biosens. Bioelectron., 10, 553-567.; 1: Stenger D A, Hickman J J, Bateman K E, Ravenscroft M S, Ma W, Pancrazio J J, Shaffer K, Schaffner A E, Cribbs D H, Cotman C W. Related Articles Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons.J Neurosci Methods. 1998 Aug. 1;82(2):167-73.) and with single neurons (e.g. LeMasson, G., E. Marder and L. F. Abbott (1993) Activity-dependent regulation of conductances in model neurons. Science 259, 1915-7.; Marder, E. and L. F. Abbott (1995) Theory in motion. Curr Opin Neurobiol 5, 832-40.; Schizas, C. N. and C. S. Pattichis (1997) Learning systems in biosignal analysis. Biosystems 41, 105-25.) are being studied using dual patch-clamp electrophysiology and imaging systems. Many others have proposed “in silica” models of intracellular function as precursors to programming cells for biological computation. See, e.g., U.S. Pat. No. 5,648,926 to Douglas et al., the contents of which are incorporated herein by reference. This application describes a hybrid system to elucidate cellular information in an efficient manner. There is a need to combine new algorithms to reproduce physiological conditions found in human, or modeled using data obtained with C. elegans and Drosophila that combines speed and utility of silicon systems and the relevance of cellular physiology to create new biological/non-biological high throughput assays.
With the increased capacity to uncover, isolate, discover, or create new substances (or even finding new uses for old substances), including new genetic materials, the need for a “functional assay,” which provides some idea of the potential effects, roles, or functions of the test substance, is even more acute. One can look at function from the standpoint of what is the function or role of molecules, compounds and proteins in an organism. One can also look at function from the standpoint of how collections of molecules, compounds and proteins create or affect pathways that are combined to comprise functional categories in a cell such as energy metabolism, extracellular signaling, transcription, or protein synthesis. These pathways underlie the processes and functions that maintain a cell and their identification helps to establish the identity and role or “cellular function” of the test substance in a higher organism. Particular groups of cells having unique “cellular functions” are intermixed in an organized fashion to create a higher organism such as an animal.
Traditionally, it has been difficult to assay the effect of a compound or protein on a cell's internal “functional categories” without observing the whole organism over a period of time. Many assays have been developed to gain information before resorting to experiments at the whole organism level, with mixed results. In vitro biochemical assays attempt to reproduced some of these pathways outside the cell, but such assays lack the interactions with the myriad of other pathways in the cell.
Fluorescence probes, microsensors and electrophysiological recordings have supplied a wealth of information but suffer from many drawbacks. Patch-clamp electrophysiological recordings can provide acute measurements but the experimental conditions lead to cell death. This drawback also applies to most fluorescent probes, which can cause toxic effects through photobleaching. Thus, a need exists in drug discovery, functional genomics and basic science for an assay that provides information and data about molecules, compounds and proteins (or their genes) as these substances interact non-invasively with a living cell and its various cellular pathways or functional categories over a period of time. Preferably, information and data about the various cellular pathways or functional categories, which are affected, are obtained from such an assay. More preferably, it would be desirable if such information and data could be captured electrophysiologically. If one can combine such features with simple sample preparation and if such an assay allows many conditions to be tested quickly on a statistically relevant number of cells, then one will have provided a very useful assay having a high throughput cellular functional capacity and which can provide much of the information that formerly could only be obtained through experiments at the whole organism level or by extensive and time consuming experimentation. A wide variety of biological compounds affect cellular functions. Marder has shown that greater than 40 biochemicals are involved in just the communication between neurons in the lobster digestive system. (Marder, E. and L. F. Abbott (1995) Theory in motion. Curr Opin Neurobiol 5, 832-40.) Many of these biochemicals are specific for ion channels, but many more act through receptors. Similar information for other systems about the interaction of known biochemicals and cellular processes or pathways can also be gleened from any neuroscience text. In addition, there is a wealth of clinical and epidemiological data that shows how a whole host of compounds affect cellular function, for example.
By indirect reference, the applicant believes that a large number of these biochemicals must affect the membrane potential of an affected cell in some way. For example, some compounds (such as saxitoxin) operate by inhibition of the sodium ion channel. Others, such as tetraethylammonium chloride (TEA) operate by acting on the potassium channel. Still other compounds activate intracellular cascades leading to calcium mobilization and specific gene activation. Hence, the applicant describes herein, systems, devices and methods that exploit the effects of biochemicals on inter alia the membrane potential. Accordingly, the applicant has discovered that one can characterize the changes in an action potential obtained from an electrically active cell following the addition of specific biochemical compounds or “triggers” to such electrically active cells (e.g., neuronal cells) using planer microelectrodes that enables ellucidation of the cellular function relevant to drug discovery or functional genomics. It should be noted that a particular embodiment of the changes in a membrane potential are the changes that can be observed in an action potential. Hence, an electrically active cell is one that exhibits perceptible (measurable) changes in its membrane potential, more preferably, one that exhibits perceptible changes in its action potential.
Examples of biochemicals of interest include, but are not limited to, those that elicit changes in signals via the following mechanisms: (a) phosphatidylinositol turn-over; (b) calcium mobilization; (c) phosphorylation of intracellular protein messengers; (d) ion channel blockers (Na+, K+, Ca2+, etc.); and (e) cAMP formation. Biochemicals can also be selected for their inhibitory properties on specific pathways, such as neurotransmission inhibitors and protein synthesis inhibitors. Some of these compounds have been shown to affect the membrane potential and other individual ion channels.
Surface modification technology utilizing Self Assembled Monolayers (SAMs) is a known process for preparing a modifying layer composed of organic molecules, which can spontaneously form strong interactions or covalent bonds with reactive groups on an exposed surface. The utilization of SAMs for modifying surfaces has been demonstrated on electronic materials such as silicon dioxide, biodegradable polymers and other polymers such as Teflon. A large variety of functional groups or combination of functional groups can be located on the terminus opposite the attachment point of a SAM, and the chemical composition can be manipulated to systematically vary the surface free energy. Biological cells can attach to, and proliferate on SAMs, and SAMs can be used to pattern a surface. SAMs are also useful to set as templates for the patterning of biomolecules, especially antibodies. SAMs thus can prove to be an ideal tool for the design of artificial surfaces for the tailoring of cellular interactions.
Metal microelectrodes surrounded by an insulator can be used to record the electrical activity of cells extracellularly. The applicant has surmised that if the interface can be tailored to keep the cells on the microelectrodes, a viable system can be created for high throughput cell assays. The applicant believes that this type of system can be fabricated by taking advantage of previous work involving orthogonal self-assembly on two different metals (see, e.g., Hickman, J. J., Laibinis, P. E., Auerbach, D. I., Zou, C., Gardner, T. J., Whitesides, G. M., and Wrighton, M. S. (1992). Toward orthogonal self-assembly of redox active molecules on Pt and Au: Selective reaction of disulfide with Au and isonitrile with Pt. Langmuir 8: 357.) and on a surface composed of a metal and an insulator coating region. See, also, U.S. Pat. No. 5,223,117 to Wrighton et al., the contents of which are incorporated herein by reference.
Surface analysis techniques have been applied to analyze cell culture surfaces both before and after culture and to relate the quantitative and qualitative results to cell morphology and survival. (See, e.g., Schaffner, A., Barker, J. L., Stenger, D. A., and Hickman, J. (1995). Investigation of the factors necessary for growth of hippocampal neurons in a defined system. J. Neurosci. Methods, 62, 111-119.) Previous studies by others have also correlated cell behavior to the initially quantified properties of the culture surface, i.e., prior to the addition of cells. Many components of the culture medium adsorb onto the surface, and cells secrete substances that comprise an extracellular matrix (ECM), as well as soluble molecules. Many of these biomolecules potentially are the source of the cell behavior monitored and can be a valuable source of information.
One problem encountered using a cell line as the sensor element is that cell lines (e.g., NG108-15, which is derived from a glioma x neuroblastoma) have an inherently unstable genome. The applicant considers primary cells to be very relevant to the present system because it is presumed that such cells more closely approximate in vivo systems than tumor-derived cell lines; however, primary cells tend to be difficult to culture and are inhomogeneous. A possible solution to these drawbacks involves the utilization of clonal cell lines derived from CNS stem cells. Thus, a preferred cell having a stable long-lived phenotype is one derived from a stem cell. In the present invention, each individual cell becomes a unique assay element with the cells localized on individual microelectrodes. Statistics can be performed on a reproducible population in response to a compound that is introduced into the media. Further we will apply system level algorithms to enable the reproduction or representation of relevant physiological states or reproductions of known assays employed by pharmaceutical or other biotechnology companies.
Hence, the present invention hopes to provide an assay of cellular function, using “functional categories” within the cell as defined, for example, by Riley, M. (1993). Functions of gene products of Escherichia coli. Microbiol. Rev. 57, 862-952. The present system is validated by taking known biochemicals with known functions and monitoring the changes in electrical potential upon introduction of the known biochemicals in the media. The present invention and its broadly applicable techniques would add a new paradigm in molecular function analysis, including gene function analysis. It is also possible to map cells in varying stages of development, as the present techniques can be applied using embryonic cells. Particularly useful cells include CNS cells, but the present approach can be used on any cell type that permits the monitoring of electrical changes in the membrane potential. It is hoped that a clear need for the present invention has been established by the discussion presented herein.